Summary of Study ST002322

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001488. The data can be accessed directly via it's Project DOI: 10.21228/M8CT5G This work is supported by NIH grant, U2C- DK119886.

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Study IDST002322
Study TitleMetabolomics study comparing SCAP KO and WT B cells
Study TypePurified mouse B cells, stimulated ex vivo
Study SummarySplenic B cells isolated from SCAP+/+ CD19-Cre (WT) and SCAPfl/fl CD19-Cre (KO) mice were stimulated with LPS or anti-CD40 for 24 and 48 hours. Cells were then analyzed by metabolomics. Metabolomics reveals global metabolic changes in SCAP deficient B cells.
Institute
Indiana University School of Medicine
Last NameLuo
First NameWei
Address950 W Walnut Street - R2 E304
Emailwl47@iu.edu
Phone3172748042
Submit Date2022-10-19
Analysis Type DetailLC-MS
Release Date2022-11-18
Release Version1
Wei Luo Wei Luo
https://dx.doi.org/10.21228/M8CT5G
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001488
Project DOI:doi: 10.21228/M8CT5G
Project Title:Metabolomics reveals global metabolic changes between WT and SCAP deficient B cells
Project Type:Purified mouse B cells, stimulated in vitro
Project Summary:Cellular metabolism regulates almost all critical cellular activities including cell growth, proliferation, energy homeostasis and signaling transduction. SCAP controls SREBP signaling, an important pathway regulating cellular lipid biosynthesis. SCAP KO B cells have multiple defects and cannot form germinal centers. To test whether these defects of SCAP KO B cells are associated with an altered global metabolic state. We stimulated B cells isolated from SCAPfl/fl CD19Cre/+ mice (KO) and SCAP+/+ CD19Cre/+ mice (WT) with ⍺-CD40 or LPS for 24 and 48 hours, followed by targeted metabolomics analysis to evaluate global metabolic changes. We found that the metabolic profiles of unstimulated SCAP KO and WT B cells were largely overlapping, corroborating our findings that SCAP deficiency did not affect B cell development or their metabolic profile at steady state. In contrast, after stimulation with either LPS or ⍺-CD40, SCAP KO and WT B cells were clearly separated at 24 hours, with an even more pronounced separation at 48 hours. SREBP signaling can regulated the metabolism of many ceramides and sphingolipids. For example, lactosyl-N-palmitoyl-sphingosine (d18:1/16:0), one of the lactosylceramides (LacCer), is highly accumulated in SCAP KO B cells activated by either ⍺-CD40 or LPS. Strikingly, besides lipid metabolism, SCAP deficiency also alters many other metabolites that belong to different metabolic pathways such as energy, amino acids, peptide, cofactors and vitamins, nucleotide, carbohydrate, and xenobiotics. Taken together, the metabolomics analysis revealed previously unrecognized roles of SREBP signaling in regulating multiple cellular activities in activated B cells.
Institute:Indiana University
Last Name:Luo
First Name:Wei
Address:950 W Walnut Street - R2 E304, Indianapolis, IN, 46202, USA
Email:wl47@iu.edu
Phone:3172748042

Subject:

Subject ID:SU002408
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:SCAP fl/fl CD19-Cre (KO) and SCAP+/+ CD19-Cre (WT)
Cell Strain Details:Splenic B cell
Species Group:Mammals

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype TREATMENT TIME_POINT
SA227408KO_CD40_24hr_4SCAP KO anti-CD40 24hr
SA227409KO_CD40_24hr_2SCAP KO anti-CD40 24hr
SA227410KO_CD40_24hr_3SCAP KO anti-CD40 24hr
SA227411KO_CD40_24hr_1SCAP KO anti-CD40 24hr
SA227412KO_CD40_48hr_1SCAP KO anti-CD40 48hr
SA227413KO_CD40_48hr_2SCAP KO anti-CD40 48hr
SA227414KO_CD40_48hr_3SCAP KO anti-CD40 48hr
SA227415KO_CD40_48hr_4SCAP KO anti-CD40 48hr
SA227396KO_0hr_2SCAP KO Freshly isolated (No treatment) 0hr
SA227397KO_0hr_3SCAP KO Freshly isolated (No treatment) 0hr
SA227398KO_0hr_1SCAP KO Freshly isolated (No treatment) 0hr
SA227399KO_0hr_4SCAP KO Freshly isolated (No treatment) 0hr
SA227400KO_LPS_24hr_3SCAP KO LPS 24hr
SA227401KO_LPS_24hr_2SCAP KO LPS 24hr
SA227402KO_LPS_24hr_4SCAP KO LPS 24hr
SA227403KO_LPS_24hr_1SCAP KO LPS 24hr
SA227404KO_LPS_48hr_3SCAP KO LPS 48hr
SA227405KO_LPS_48hr_2SCAP KO LPS 48hr
SA227406KO_LPS_48hr_4SCAP KO LPS 48hr
SA227407KO_LPS_48hr_1SCAP KO LPS 48hr
SA227428WT_CD40_24hr_2WT anti-CD40 24hr
SA227429WT_CD40_24hr_1WT anti-CD40 24hr
SA227430WT_CD40_24hr_4WT anti-CD40 24hr
SA227431WT_CD40_24hr_3WT anti-CD40 24hr
SA227432WT_CD40_48hr_2WT anti-CD40 48hr
SA227433WT_CD40_48hr_1WT anti-CD40 48hr
SA227434WT_CD40_48hr_3WT anti-CD40 48hr
SA227435WT_CD40_48hr_4WT anti-CD40 48hr
SA227416WT_0hr_4WT Freshly isolated (No treatment) 0hr
SA227417WT_0hr_3WT Freshly isolated (No treatment) 0hr
SA227418WT_0hr_2WT Freshly isolated (No treatment) 0hr
SA227419WT_0hr_1WT Freshly isolated (No treatment) 0hr
SA227420WT_LPS_24hr_1WT LPS 24hr
SA227421WT_LPS_24hr_2WT LPS 24hr
SA227422WT_LPS_24hr_4WT LPS 24hr
SA227423WT_LPS_24hr_3WT LPS 24hr
SA227424WT_LPS_48hr_4WT LPS 48hr
SA227425WT_LPS_48hr_1WT LPS 48hr
SA227426WT_LPS_48hr_2WT LPS 48hr
SA227427WT_LPS_48hr_3WT LPS 48hr
Showing results 1 to 40 of 40

Collection:

Collection ID:CO002401
Collection Summary:Mouse splenic B cells were isolated using STEM CELL B cell isolation kit (Cat:19854)
Sample Type:B cells

Treatment:

Treatment ID:TR002420
Treatment Summary:Splenic B cells isolated from SCAP+/+ CD19-Cre (WT) and SCAPfl/fl CD19-Cre (KO) mice were stimulated with 10 micro g/ml LPS or 5 micro g/ml anti-CD40 for 24 and 48 hours. Freshly isolated untreated WT and KO B cells were used as untreated control.

Sample Preparation:

Sampleprep ID:SP002414
Sampleprep Summary:Sample preparation: Proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phases (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

Combined analysis:

Analysis ID AN003789
Analysis type MS
Chromatography type Reversed phase/HILIC
Chromatography system Waters ACQUITY ultra-performance liquid chromatography (UPLC)
Column Waters UPLC BEH C18 (100 x 2.1mm,1.7um)/Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Exactive Plus Orbitrap
Ion Mode UNSPECIFIED
Units Normalized AUC

Chromatography:

Chromatography ID:CH002802
Methods Filename:B_cell_MS_protocol.docx
Instrument Name:Waters ACQUITY ultra-performance liquid chromatography (UPLC)
Column Name:Waters UPLC BEH C18 (100 x 2.1mm,1.7um)/Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Chromatography Type:Reversed phase/HILIC

MS:

MS ID:MS003531
Analysis ID:AN003789
Instrument Name:Thermo Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments: Analysis was performed by Metabolon. Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS): All methods utilized a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. The sample extract was dried then reconstituted in solvents compatible with each of the four methods. Each reconstitution solvent contained a series of standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion conditions, chromatographically optimized for more hydrophilic compounds. In this method, the extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 µm) using water and methanol, containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Another aliquot was also analyzed using acidic positive ion conditions; however it was chromatographically optimized for more hydrophobic compounds. In this method, the extract was gradient eluted from the same aforementioned C18 column using methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA and was operated at an overall higher organic content. Another aliquot was analyzed using basic negative ion optimized conditions using a separate dedicated C18 column. The basic extracts were gradient eluted from the column using methanol and water, however with 6.5mM Ammonium Bicarbonate at pH 8. The fourth aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1x150 mm, 1.7 µm) using a gradient consisting of water and acetonitrile with 10mM Ammonium Formate, pH 10.8. The MS analysis alternated between MS and data-dependent MSn scans using dynamic exclusion. The scan range varied slightly between methods but covered 70-1000 m/z.
Ion Mode:UNSPECIFIED
Analysis Protocol File:B_cell_MS_protocol.docx
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